This invention relates generally to the in vitro synthesis of peptides and, more particularly, to the synthesis of nascent peptides on Escherichia coli ribosomes using synthetic tRNA species.
Scientists have for a long time been trying to develop a methodology that will make it possible to efficiently modify and produce proteins outside living cells. One technical component of this endeavor is the modification of tRNAs with altered anticodon or recognition sites for enzymatic aminoacylation. The modified tRNAs make it relatively simple to substitute different amino acids at a target site of a protein by generating a single new codon which can be read by each of a series of modified tRNAs.
By using a series of modified tRNAs, one each for a different ammo acid, the specific amino acid can be incorporated into the protein at the target site(s) of interest. Since under most circumstances only one protein is synthesized in any system, the effect of the amino acid substitution on the activity of the polypeptide can be measured directly without producing a clone for each amino acid substitution and without the problems frequently associated with expression and subsequent testing of a protein produced in intact cells.
Ultimately, as the ability to predict the tertiary structure of a protein from its amino acid sequence is refined, it will be possible to produce totally designed proteins using mRNA that contain only 20 codons, one for each of the 20 naturally occurring amino acids for which there is a codon in the universal genetic code. Amino acids also can be incorporated into one or more specific sites in the protein by using modified tRNAs containing anticodons for some of the unused 41 codons. Further, by using chemical, rather than enzymatic aminoacylation of the modified tRNAs, it will be possible to incorporate amino acids that have no naturally occurring counterpart into these "artificial proteins." This will make it possible to design, test, and produce artificial enzymes that use catalytic mechanisms and substrates that do not occur in living organisms.
The acylation of tRNA with a specific amino acid may be carried out by either chemical or enzymatic reactions, the latter using aminoacyl-tRNA synthetases (AS). Procedures for chemical aminoacylation of tRNAs are known. S. A. Robertson et al. (1989) Nucl. Acid Res. 17:9649-9660; M. Hagen et al. (1988) J. Org. Chem. 53:5040-5045.
In living systems, an AS must recognize with high specificity both the amino acid and the tRNA to be aminoacylated. Recognition of a tRNA species by the AS depends on specific structural features in the tRNA. There is no one single set of structural features in different tRNAs that specify their recognition by their cognate AS. C. de Duve (1988) Nature 333:117-118. Many of the structural features for tRNA identity appear not to be strictly conserved for a specific tRNA species in different organisms. Considerable variation in the specificity for aminoacylation of synthetic tRANs has been found with ASs from E. coli, yeast and rabbit reticulocytes and for conditions (salt and spermine or spermidine concentration, temperature) under which aminoacylation is carried out. Some principles for tRNA recognition by cognate AS have emerged. Some AS appear to have strict requirements for all of the anticodon, while others recognize only part of the anticodon. Met-tRNA AS appear to be highly dependent on the CAU anticodon (G. Ghosh et al. (1990) Biochem. 29:2220-2225); ASs for other tRNAs may require the second or third nucleotide of the anticodon.
ASs for other amino acids appear to recognize features of tRNA that are independent of the anticodon, but respond to positive, and in some cases negative, recognition sites elsewhere in the tRNA structure. The result of Schimmel and Hou demonstrate that G3-C70 base pair in the amino acid stem of tRNA.sup.Ala is the primary recognition cite for the AlatRNA AS. Y. M. Hou et al. (1988) Nature 333:140-145.
It is now potentially possible to design and produce synthetic tRNAs in vitro that are enzymatically aminoacylated and can be used in cell-free translation systems. These synthetic tRNAs may be of considerable value in elucidating ribosome function particularly with respect to movement of the nascent peptide within the ribosome and its folding into an active confirmation. The precise position and confirmation of nascent peptide as they are formed in ribosomes remain unknown.